Heme Chemistry

Gandham. Rajeev

Hemoglobin

Hemoglobin is red blood pigment, found in erythrocytes

It is a chromoprotein, containing heme as the prosthetic

group & globin as the protein part-apoprotein

Heme containing proteins are characteristic of aerobic

organisms

Normal levels

Adult male:14 to 16 gm%

Female:13 to 15 gm%

Hemoglobin is a tetrameric protein & molecular weight 64,450

Approximately 6.25 gm of Hb are produced & destroyed in

body each day

The basic protein “globin” varies from species to species in

its amino acid composition & sequence, and is responsible for

species-specificity

Polypeptide chains of globin of adult Hb, contain high

content of ‘histidine’ & ‘lysine’ & small amount of isoleucine

Functions of Hemoglobin

Delivery of O2 from lungs to the tissues

Transport of CO2 & protons from tissues to lungs for

excretion

Heme is present in Myoglobin, Cytochromes, Peroxidase,

Catalase, Tryptophan pyrrolase & Nitric oxide synthase

In cytochromes, oxidation & reduction of iron is essential

for their biological function in ETC

Structure of Globin

Globin consists of 4 polypeptide chains

Adult Hb is made up of 2α-chains & 2β-chains (α2 β2)

Each α -chain contains 141 AAs & β-chain contains 146 AAs.

HbA1, has a total of 574 amino acids

The four subunits of hemoglobin are held together by non-

covalent interactions - hydrophobic, ionic & hydrogen bonds.

Each subunit contains a heme group

Structure of Heme

Heme is a Fe-porphyrin compound

Porphyrins are cyclic compounds formed by fusion of 4

pyrrole rings linked by methenyl (=CH–) bridges

Since an atom of iron is present, heme is a

ferroprotoporphyrin.

The pyrrole rings are named as I, II, III, IV and the

bridges as alpha, beta, gamma and delta

Porphyrin ring

Heme contains a porphyrin molecule, protoporphyrin lX,

with iron at its center

Protoporphyrin lX consists of four pyrrole rings to which

four methyl, two propionyl & two vinyl groups are

attached

Structure of heme

Formation of Heme Pockets

Each polypeptide chain contains a ‘heme pocket’

Hb molecule & its sub-units contains hydrophobic amino

acids internally & hydrophilic amino acids on their surfaces

The heme pockets of α-subunits are of size, adequate for

entry of O2 molecule, but the entry of O2 into the heme-

pockets of β-subunits is blocked by valine.

Differences between α & β-chains of adult normal Hb

α-Subunit β-Subunit

Molecular weight 15126 15866

Total amino acids 141 146

C-terminal amino acid Arginine Histidine

N-terminal amino acid Val-Leu Val-His-Leu

α-Helices 7 8

Heme-pocket Adequate for entry of one molecule of O2

Entry of O2 in heme-pocket is blocked by valine

Other forms of Haemoglobin

Hb-A1:

Normal adult Hb, commonly called Hb-A, consists of 2 α -

& 2 β chains (α2β2)

It is approximately 90% of total haemoglobin

Hb-F:

It is a human foetal haemoglobin

Consisting of α2γ2

Differentiation of Hb-A from Hb-F

Hb-A Hb-F

Two α & two β chains Two α & two γ chains

Denatured by alkali Resistant to alkali denaturation

At pH 8.9 Hb-A moves ahead of Hb-F Hb-F moves behind Hb-A

2,3-BPG content is high 2,3-BPG content is low

Affinity of O2 is less Affinity to O2 is more

Delivery power of O2 more (unloading) Delivery power of O2 is decreased

Concentration at birth-Hb-A=85% 15%

Hb-F disappears by end of first year, persistence of Hb-F after one year is pathological

Hb-A2:

It is a minor component of normal adult Hb.

It contains two α & two δ-chains α2 δ2

It is approximately-2.5%

Electrophoretically, it is a slowly migrating fraction

Hb-A3:

It amounts for 3 to 10% of total haemoglobin

It is a fast moving fraction

Normal major types of haemoglobin

Type Composition % of total haemoglobin

HbA1 α2β2 90%

HbA2 α2 δ2 <5%

HbF α2γ2 <2%

HbA1c α2β2-glucose <5%

Hb-A1c (Glycosylated Hb):

It is formed by covalent binding of glucose to haemoglobin

Its normal range is 3 to 6%

Its levels are increased in diabetes mellitus

Chemistry:

The amino acid sequence of HbA1c is exactly same as that

of HbA1

The attachment of 1-amino 1-deoxy fructose to the –NH2

terminal of valine of β-chain of HbA1

Addition of sugar moiety to valine occurs non-enzymatically,

either by addition of glucose directly to the protein.

Diagnostic importance of HbA1c:

The rate of synthesis of HbA1c is directly related to the

exposure of RBC to glucose

The concentration of HbA1c serves as an indication of blood

glucose concentration over a period

HbA1c reflects the mean blood glucose level over 3 months

period prior to its measurement

In diabetes, HbA1c is elevated to as high as 15%

Determination of HbA1c is used for monitoring of diabetes

If the HbA1c concentration is <7%, the diabetic patient is

considered to be in good control

Myoglobin

Myoglobin (Mb) is monomeric O2 binding hemoprotein

Found in heart and skeletal muscle.

lt has single polypeptide (153 A.As) chain with heme moiety.

Myoglobin (mol. wt. 17,000) structurally resembles the

individual subunits of hemoglobin molecule

Myoglobin functions as a reservoir for oxygen.

It serves as oxygen carrier that promotes the transport of

oxygen to the rapidly respiring muscle cells

Binding of O2 to haemoglobin

One molecule of Hb can bind with four molecules of O2.

Myoglobin (with one heme) which can bind with only one

molecule of oxygen.

In other words, each heme moiety can bind with one O2.

Transport of O2 by haemoglobin

It can transport large quantities of oxygen

It can take up and release oxygen at appropriate partial

pressures

It is a powerful buffer.

Oxygen Dissociation Curve (ODC)

The binding ability of hemoglobin with oxygen at

physiological pO2 (partial pressure of oxygen) is shown

by the oxygen dissociation curve (ODC)

At the oxygen tension in the pulmonary alveoli, the Hb is

97% saturated with oxygen.

Oxygen dissociation curve (ODC)

Factors affecting oxygen dissociation curve

Heme-heme Interaction & Cooperativity:

The oxygen dissociation curve (ODC) is sigmoid shape.

The binding of O2 to one heme residue increases the affinity

of remaining heme residues for O2.

Thus the affinity of Hb for the last O2 is about 100 times

greater than the binding of the first O2 to Hb.

This is called positive cooperativity

Release of O2 from one heme facilitates the release of O2

from others.

The quaternary structure of oxy-Hb is described as R

(relaxed) form; & deoxy- Hb is T (tight) form.

2α +2β(Deoxy-Hb – T-form)

2α,β(Oxy-Hb – R-form)

T and R forms of hemoglobin

The four subunits (α2β2) of hemoglobin are held together

by weak forces.

The relative position of these subunits is different in

oxyhemoglobin compared to deoxyhemoglobin.

T-form of Hb:

The deoxy form of Hb exists in T or taut (tense) form.

The H & ionic bonds limit the movement of monomers.

The T-form of Hb has low oxygen affinity.

R-form of Hb

The binding of O2 destabilizes some of the hydrogen &

ionic bonds particularly between αβ dimers.

This results in a relaxed form or R-form of Hb

Therefore, the R-form has high oxygen affinity.

Transport of CO2 by hemoglobin

ln aerobic metabolism, for every molecule of O2 utilized,

one molecule of CO2 is liberated.

Hemoglobin actively participates in the transport of CO2

from the tissues to the lungs.

About 15% of CO2 carried in blood directly binds with Hb.

The rest of the tissue CO2 is transported as bicarbonate

(HCO3).

CO2 molecules are bound to the uncharged α-amino acids of

hemoglobin to form carbamyl hemoglobin.

The oxyHb can bind 0.15 moles CO2/mole heme, whereas

deoxyHb can bind 0.40 moles CO2/mole heme.

The binding of CO2 stabilizes the T (taut) form of hemoglobin

structure, resulting in decreased O2 affinity for Hb.

Hemoglobin also helps in the transport of CO2 as

bicarbonate

CO2 enters the blood from tissues, the enzyme carhonic

anhydrase present in erythrocytes catalyses the formation

of carbonic acid (H2CO3).

Bicarbonate (HCO3-) & proton (H+) are released on

dissociation of carbonic acid

Hb acts as a buffer & immediately binds with protons

Every 2 protons bound to Hb, 4 oxygen molecules are

released to the tissues.

In the lungs, binding of O2 to Hb results in the release of

protons.

The bicarbonate & protons combine to form carbonic acid.

Acted upon by carbonic anhydrase to release CO2, which is

exhaled

The Bohr Effect

The binding of O2 to hemoglobin decreases with increasing

H+ concentration (lower pH) or when the hemoglobin is

exposed to increased partial pressure of CO2 (pCO2).

This phenomenon is known as Bohr effect.

It is due to a change in the binding affinity of O2 to

hemoglobin

Bohr effect causes a shift in the oxygen dissociation curve to

the right

Bohr effect is primarily responsible for the release of O2

from the oxyhemoglobin to the tissue.

This is because of increased pCO2 & decreased pH in the

actively metabolizing cells

Binding of CO2 forces the release of O2.

When carbonic acid ionizes, the intracellular pH falls.

The affinity of Hb for O2 is decreased & O2 is unloaded to

the tissues.

CO2+H

2O H

2CO

3H

++HCO

3

Carbonic anhydrase

The Chloride Shift

When CO2 is taken up, the HCO3 ¯ concentration within the

cell increases.

This would diffuse out into the plasma.

Chloride ions from the plasma enter into cell to establish

electrical neutrality.

This is called chloride shift or Hamburger effect.

RBCs are slightly bulged due to the increased chloride ions

Chloride shift in tissues

When the blood reaches the lungs, the reverse reaction takes

place.

The deoxyhemoglobin liberates protons (H+).

These H+ combine with HCO3 – to form H2CO3.

H2CO3 dissociated to CO2 & H2O by the carbonic anhydrase.

The CO2 is expelled.

HCO3 – binds H+, more HCO3– from plasma enters the cell &

Cl– gets out (reversal of chloride shift)

Chloride shift in lungs

Effect of 2,3-BPG

2,3-Bisphosphoglycerate is the most abundant organic

phosphate in the erythrocyte.

The 2,3-BPG is produced from 1,3-BPG, an intermediate of

glycolytic pathway

This short pathway, referred to as Rapaport-Leubering cycle

The 2,3-BPG, binds to deoxy-Hb (and not to oxyhemoglobin)

& decreases the O2 affinity to Hb & stabilizes the T

conformation.

As oxygen is added, salt bridges are successively broken and finally 2,3-BPG is expelled. Simultaneously the T (taught) confirmation of deoxy-Hb is changed into R(relaxed) confirmation of oxy-Hb. Blue circle represents 2,3-bisphosphoglycerate (BPG)

When the T form reverts to the R conformation, the 2,3-BPG

is ejected.

The reduced affinity of O2 to Hb facilitates the release o f O2

at the partial pressure found in the tissues.

2,3-BPC shifts the oxygen dissociation curve to the right

The high oxygen affinity of fetal blood (HbF) is due to the

inability of gamma chains to bind 2,3-BPG.

Mechanism of action of 2,3-BPG

One molecule of 2,3-BPG binds with one molecule (tetramer)

of deoxyhemoglobin in the central cavity of the four

subunits.

This central pocket has positively charged (e.g. histidine,

lysine) two β-globin chains.

lonic bonds (salt bridges) are formed between the positively

charged amino acids (of β-globins) with the negatively

charged phosphate groups of 2,3-BPG

Binding of 2,3-BPG stabilizes the deoxygenated

hemoglobin (T-form) by crosslinking the β -chains

On oxygenation of hemoglobin, 2,3-BPG is expelled from

the pocket and the oxyhemoglobin attains the R-form of

structure

Clinical significance of 2,3-BPG

ln hypoxia:

The 2,3-BPG in erythrocytes is elevated in chronic hypoxic

conditions associated with difficulty in O2 supply.

These include adaptation to high altitude, obstructive

pulmonary emphysema

ln anemia:

2,3-BPC levels are increased in severe anemia in order to

cope up with the oxygen demands of the body.

This is an adaptation to supply as much O2 as possible to

the tissue, despite the low hemoglobin levels.

In blood transfusion:

Storage of blood in acid citrate-dextrose medium results

in the decreased concentration of 2,3-BPG.

Such blood when transfused fails to supply O2 to the

tissues immediately.

Addition of inosine (hypoxanthine-ribose) to the stored

blood prevents the decrease of 2,3-BPG.

The ribose moiety of inosine gets phosphorylated & enters

the hexose monophosphate pathway and finally gets

converted to 2.3-BPG

References

Text book of Biochemistry – U Satyanarayana

Text book of Biochemistry – DM Vasudevan

Text book of Biochemistry – MN Chatterjea

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